PROJECT 3: SUMMARY Glioblastoma (GBM), the most aggressive and prevalent manifestation of malignant glioma, is characterized by resistance to extant therapeutic modalities, and exhibit a neurologically debilitating course culminating in death, often within 14 months after diagnosis. Among the critical challenges for improving treatment outcomes for GBM patients are the identification and characterization of new drug targets to overcome the notorious therapy resistance of GBM, and the development of drug delivery platforms to target undruggable genetic lesions. Restoration of p53 activity represents an attractive therapeutic strategy for the treatment of GBM, as ~65% of primary GBM patients express wildtype but functionally suppressed p53. Amplification and overexpression of the atypical Bcl2 family protein Bcl2L12 (Bcl2-Like-12) compromises p53 function by blocking the transcriptional activity of p53. To inhibit Bcl2L12 function, we propose to use novel RNAi-based nanoconjugates, termed Spherical Nucleic Acids (SNAs) to neutralize Bcl2L12 expression in established GBM. We have found that Bcl2L12-targeting SNAs (siBcl2L12-SNAs) are able to traverse cellular membranes including the blood-brain-barrier. We established that siBcl2L12-SNAs do not require the use of toxic auxiliary reagents and accumulate effectively in GBM tumor cells upon crossing the blood-brain/blood-tumor barrier in intracerebral GBM xenografts following systemic administration of the SNAs. SNAs exhibit stability in physiological environments, provoke robust intratumoral Bcl2L12 mRNA and protein knockdown resulting in p53 reactivation, and slow tumor growth in GBM patient derived xenograft (PDX) models. Here, we will further investigate the hypothesis that Bcl2L12 ablation by SNA treatment increases p53 tumor suppression, slows GBM progression, and can be combined with conventional genotoxic therapies as well as with targeted therapeutics for improved suppression of tumor growth, and possibly for causing tumor regression. In Aim 1, we will determine siBcl2L12 treatment effect in patient-derived glioma-initiating cells (GICs) in vitro, and in PDX models in vivo, as monotherapy and in combination with radiation therapy (RT). In Aim 2, using both PDX models for newly diagnosed and recurrent tumor, together with syngeneic, immunocompetent mouse models, we will combine siBcl2L12 with cytotoxic and p53-activating chemotherapeutic drugs, i.e., the DNA alkylator temozolomide and the MDM2 inhibitor RG7388, respectively. Aim 3 proposes a phase 0 clinical trial of siBcl2L12-SNAs, to determine SNA pharmacokinetics, biodistribution, and ability to downregulate GBM- associated Bcl2L12 mRNA and protein. The results of this proposal will provide an in-depth characterization of the Bcl2L12 oncoprotein as an actionable GBM oncoprotein, and will pave the way to successfully implement SNA-mediated, multi-modal p53 reactivation as a therapeutic approach to incorporate in clinical practice.